Spectrophotometers are laboratory tools for analyzing or testing liquid samples of, for example, chemical solutions or mixtures, biologic materials, biochemical materials, biochemical reactions, and the like. Typically, two types of spectrophotometers are available: the vertical light beam spectrophotometer, in which the analyzing light beam is transmitted in the vertical direction through a sample disposed in a horizontal plane; and the horizontal light beam spectrophotometer in which the analyzing beam is transmitted in the horizontal direction through a sample disposed in a vertical plane.
Most conventional spectrophotometers employ a horizontal light beam that traverses the liquid sample horizontally so as to avoid passing through the liquid-gas interface that is typically above the sample. With such horizontal light beam photometers, the geometry and optical pathlength within the sample is fixed for any given cuvette. For visible and ultraviolet light absorption measurements, for example, cuvettes customarily have a 1 cm pathlength. Cuvettes with pathlengths between 0.1 cm and 10 cm are also common, however.
Vertical light beam spectrophotometers also measure light absorption. In vertical light beam spectrophotometers, however, the light beam typically passes only through one wall of the sample-retaining device, through the sample, and through the interface between the sample a surrounding gas atmosphere (which is usually air).
The latter liquid-gas interface, the meniscus, is usually curved. The specific shape of the meniscus depends upon the interactions between the liquid sample and the gas and the sidewalls of the sample-retaining device. Depending upon the design of a particular vertical light beam spectrophotometer the light beam may traverse the meniscus either before or alter passing through the sample. In either case, the optical pathlength through the sample is not a constant value. Instead, the optical pathlength is related to the sample volume and the meniscus shape. The nature of the sample, the sample-retaining device surfaces, and the gas each contributes to the shape of the meniscus, quantitatively affecting the optical pathlength through the sample.
Vertical light beam spectrophotometry has become a popular technique, despite the disadvantage of not having a fixed optical pathlength through the sample. This popularity stems from the fact that the optical characteristics of multiple samples may be analyzed with a vertical-beam photometer in a small period of time. Typically, vertical light beam spectrophotometers monitor the optical characteristics of samples disposed in the wells of, for example, 96-well multi-assay plates. The optical characteristics, such as light absorption or light scattering, of the samples contained within each well of such multi-assay plates may be monitored in a few seconds. Vertical light beam spectrophotometers also allow repetitive measurements of multiple samples to be made with short intervals between each of a series of measurements.
The use of vertical light beam spectrophotometers in clinical assays is therefore important. These vertical light beam spectrophotometers are calibrated, and the calibrations are verified by the manufacturer. However, it is both necessary and good practice to routinely validate the performance of these instruments in the course of clinical practice, and may in fact be required by regulatory agencies overseeing such practice.
There exists a number of U.S. patents directed to verifying the calibration of vertical light beam spectrophotometers, including U.S. Pat. No. 5,258,308 issued to Freeman, et. al., entitled “Method, kit and apparatus for verifying calibration and linearity of vertical photometers,” (“Freeman”). Freeman describes a method and means of verifying the calibration of vertical light beam spectrophotometers comprising pre-dispensed dye check strips which provide reproducible standards by which the functioning of the spectrophotometer can be ascertained. Each strip contains a plurality of microtiter wells containing a dried dye material which when reconstituted can be read in the various models of photometers and spectrophotometers to assess instrument performance.
U.S. Pat. No. 5,963,318 issued to Held, entitled “Method of and apparatus for performing fixed pathlength vertical photometry,” (“Held”) describes a system for performing vertical light beam spectrophotometric determinations using a vessel which has an upper transparent surface and a lower transparent surface which are spaced apart to define a known fixed pathlength through which a light beam is transmitted. The vessel also includes a portion disposed above the upper surface which may be a spout having an opening for allowing the introduction of substances into the containment portion.
Like Held, U.S. Pat. No. 6,074,614 issued to Hafeman, et. al., entitled “Multi-assay plate cover for elimination of meniscus.” (“Hafeman”) also describes a vessel having a constant pathlength, where the vessel comprises a flat top side and a flat bottom side, the bottom side having solid cylindrical projections of equal length extending downwardly from the flat bottom side, wherein each cylindrical projection is centered about the optical axis passing through a corresponding sample well of a multi-assay plate, thereby eliminating meniscus and evaporation effects.
Freeman, Held, and Hafeman each describe a specialized vessel designed exclusively for calibrating and validating a vertical light beam spectrophotometer. They illustrate how a user is limited to specialized cuvettes when using a vertical light beam spectrophotometer. Accordingly, there is a need in the art for a device and method for using, calibrating, and validating a vertical light beam spectrophotometer using conventional cuvettes.
Others have attempted to validate vertical light beam spectrophotometers with conventional cuvettes in the past. For example, as discussed in U.S. Pat. No. 7,061,608 to Bradshaw. et. al., one method for calibrating vertical light beam spectrophotometers involves the testing of reference cuvettes having samples of reference concentrations. Solutions containing different concentrations of the specific dye are first sealed in conventional cuvette. Such “reference samples” or “reference solutions” typically have a known or expected absorbance measurement for comparison by a user to a measurement obtained from the spectrophotometer to be validated. However, this general method has limitations that constrain its usefulness. Bradshaw, et. al. points out that sealed cuvettes require an expansion allowance zone including a compressible component that may be a bubble of gas (such as air) to allow for the solution to expand/contract due to thermal fluctuations. This compressible component must be held out of the light beam path when such cuvettes are placed horizontally in a vertical light beam spectrometer, or it will adversely affect the absorbance values measured. Bradshaw, et. al. discloses an enclosed calibration plate including one or more sealed reference cuvettes having specialized bubble traps used to hold the compressible component in place in the expansion allowance zone, out of the beam path near the top of the cuvette. However, an easy to use system that does not require the use of specialized cuvettes having, e.g., bubble traps is still needed.
Accordingly, the present invention is directed an adaptor plate to allow the use of conventional spectrophotometer cuvettes with a vertical light beam spectrophotometer without the need for specialized cuvettes, or bubble traps, as discussed above.